Project Summary The focus is on the mechanism by which bacterial viruses (phages) achieve destruction (lysis) of the host cell and release of the viral progeny. The Gram-negative bacterial cell has three layers: the cytoplasmic or inner membrane (IM), the cell wall or peptidoglycan (PG), and the outer membrane (OM). Recent progress has revealed that dsDNA phages that dominate the biosphere encode three types of proteins, each responsible for attacking one of the three components of the cell wall. The overall process is controlled by holins, small proteins that accumulate in the IM for typically 15 to 60 minutes, when, suddenly, they trigger to form holes. The sudden formation of these membrane holes kills the cell and instantly stops all energy metabolism, marking the end of the infection cycle. This allows another class of proteins called endolysins are able to attack the cell wall, degrading the sugar-sugar or peptide linkages. Once the PG network is destroyed, a newly discovered class of proteins, called the spanins, conducts the final step. Spanins connect the IM to the OM through the PG meshwork. Once that meshwork is destroyed, the spanins undergo a conformational change and disrupt the OM. We will test a model that the destruction of the OM is the result of membrane fusion with the IM, thus removing the last barrier to virus release. The first Aim is to characterize the spanins at the molecular and structural levels, using genetics, molecular biology, cell biology, ultrastructural microscopy, biochemistry and structural studies. The second Aim, a major collaborative effort with the co-PI Dr. J. Xiao of Johns Hopkins, is focused on using super-resolution microscopy in achieving a complete description of the lysis process in four dimensions (space and place in the cell, as well as time in the infection cycle.) These studies will further our understanding of how bacterial viruses, or phages, kill their prey and effect dispersal of their progeny. Besides illuminating many fundamental processes, this may have direct practical benefits because there is a growing consensus that phages, as natural antibacterial agents, will become an important tool in combating bacterial pathogens, which are increasingly resistant to available antibiotics.